Although a substantial amount of research has been directed to the development of sequencing methods, a limitation of current day techniques is that sequence information is obtained in units of only 400 to 600 nucleotides. For genome sequencing projects, such as sequencing the human genome, it would be inefficient to fit together such small units of sequence information. Longer units of sequence information would also be required in order to sequence through many repeated DNA sequences.
One proposed method to increase the length of sequence information is the single molecule sequencing method U.S. Pat. No. 4,962,037; Jett, J. H., et al., J. Biomolecular Structure & Dynamics, 7:301-309 (1989); Ambrose, et al., Ber. Bunseniges Phys. Chem., 97:1535 (1993)!. For the single molecule sequencing method, a DNA polymerase is used to synthesize a complementary DNA with fluorophore-labeled deoxynucleoside triphosphates (fluorophore dNTPs). Each of the four fluorophore dNTPs has a unique fluorophore tag that can be used to identify the nucleotide. A single fluorophore-labeled DNA is then immobilized in a flow cell and subjected to exonuclease digestion. A flow system carries each released fluorophore-labeled deoxynucleoside monophosphate (fluorophore dNMP) to a highly sensitive fluorescence detector capable of single molecule detection. The order of the fluorophore dNMPs detected gives the sequence. Because in vitro fluorophore-labeled DNA synthesized in this manner may be tens of thousands of nucleotides in length, this method will be useful in providing long sequence information.
The single molecule sequencing method has two primary enzymatic components. The first enzymatic component is employed in the synthesis of the complementary fluorophore-labeled DNA, synthesis being achieved by DNA polymerase-mediated incorporation of fluorophore-labeled nucleotides. The second enzymatic component is involved in digestion of the fluorophore-labeled DNA to release fluorophore dNMPs.
In principle, DNA polymerases from a variety of organisms would appear to have the potential to be used in in vitro reactions for the synthesis of complementary, fluorophore-labeled DNA. In practice, few DNA polymerases have been found to be suitable for this purpose. Synthesis of the complementary, fluorophore DNA requires first that the DNA polymerase have the ability to incorporate the fluorophore nucleotide. Second, the DNA polymerase must then be able to extend the fluorophore-labeled terminal nucleotide by addition of the next complementary fluorophore nucleotide. Incorporation of fluorophore nucleotides and extension of a fluorophore-labeled terminus are steps that are discriminated against by most DNA polymerases. A third requirement is that DNA replication must be accurate so that a faithful complementary fluorophore-labeled DNA is synthesized.
Methods for the synthesis of long fluorophore-labeled DNA can also be used to make shorter labeled DNAs, to be used as probes. DNA probes are used to identify chromosomes, locate genes and mRNA, etc. These methods can also be used to synthesize biotin-labeled DNA, DIG-labeled DNA, etc., which rely on the enzymatic incorporation into DNA of a labeled or modified nucleotide. "DIG" is the abbreviation of digoxigenin. For the biotin- and DIG-labeled DNAs, biotin- or DIG-labeled nucleotides are used; a fluorophore-dNTP is used for the synthesis of fluorophore-labeled DNA.
Another deficiency in current DNA sequencing methods is speed. The single molecule sequencing method has the potential to increase sequencing speed to 10 or more nucleotides per second U.S. Pat. No. 4,962,037; Jett, J. H., et al., J. Biomolecular Structure & Dynamics, 7:301-309 (1989)!. Another method that has the potential to increase speed is mass spectrometry Chen, C. H., et al., SPEI 2386:1322 (1995)!. Presently, a mass spectrometric method has been reported to sequence a 35-nucleotide oligomer in a few seconds. A limitation of mass spectrometry is that only short DNAs can be sequenced. Longer DNAs can be sequenced by mass spectrometry if the differences in mass between the four nucleotides can be increased. One way to increase differences in the mass of nucleotides is to use modified nucleotides, hence, synthesis of a complementary DNA with modified nucleotides may be the means to make mass spectrometry a useful sequencing method.
It is an object of the present invention to provide compositions and methods which do not suffer from all the drawbacks of the prior art.